KR100761486B1 - Phage-Dependent Superproduction of Biologically Active Protein and Peptides - Google Patents

Abstract

This invention relates to a method for enhancing the production of biologically active proteins and peptides in bacterial cells by infecting bacterial cells of the producer strain, which contain a plasmid with one or more targeted genes, with bacteriophage lambda with or without the targeted gene(s). The targeted genes encoding the biologically active proteins are under the control of a T7 polymerase promoter and the bacteria also are capable of expressing the gene for T7 RNA polymerase. The phage increases synthesis of the targeted protein and induces lysis of the producer strain cells. Super-production is achieved by the combination of the high level of expression achieved from the T7 polymerase promoter and by cultivating the producer strain cells under culture conditions that delay lytic development of the phage. The biologically active proteins and peptides subsequently accumulate in a soluble form in the culture medium as the cells of the producer strain are lysed by the phage.

Description

Phage-Dependent Superproduction of Biologically Active Protein and Peptides

TECHNICAL FIELD The present invention relates to DNA recombination techniques, and more particularly to novel methods of enhancing the production of heterologous proteins in bacterial host cells. The disclosed method includes infecting a host cell with bacteriophage λ containing a plasmid encoding a gene of interest linked to act on the T7 promoter to induce lysis of the bacterial host cell. Mass production can be achieved in selected host cells when only the plasmid has one or more replication DNA of heterologous origin or the plasmid and phage each have one or more replication DNA of heterologous origin.

Today, genetic engineering technology enables the production of microbial strains capable of producing large quantities of a variety of biologically active substances that can be important applications in medicine and industry. Generally, plasmid vectors into which heterologous genes are inserted are used to transform bacterial host cells. Other E. coli strains are often used as recipient cells. Using transformation methods with such plasmids, a variety of valuable human proteins, including insulin, γ-interferon, numerous interleukins, superoxide dismutases, plasminogen activators, tumor necrosis factor, erythropoietin, and the like, E. coli cells have been industrially processed to produce peptides. These substances are already in use in the pharmaceutical community or are undergoing other stages of clinical research.

However, plasmid technology has serious drawbacks. The desired product must be extracted from bacteria cells after biosynthesis, which is a multistep process, which is technically complex. For example, interferon extraction is subject to cell disruption, buffer solution extraction, polyethylenmenin process, illumination, precipitation with ammonium sulfate, dialysis, and centrifugation (Goeddel, EP 0043980). The need for such extraction and purification procedures not only complicates the technology of production of recombinant products, but also results in significant losses, especially in industrial mass production.

To further complicate the problem, when the cloned genes are expressed at relatively high levels, the resulting eukaryotic proteins tend to accumulate in the cytoplasm of E. coli as an insoluble population, which is often associated with cell membranes. As a result, the extraction and purification process described above is already a difficult process in itself, and requires an additional process for the extraction of insoluble objects accumulated therein.

In general, insoluble proteins are allowed to dissolve using ionic detergents such as SDS or lauryl sarcosine at high temperatures or in the presence of denaturants such as 8M urea or 6-8M guanine-hydrochloric acid.

Occasionally, the final stage of purification will be the process of renaturation and reoxidation of the dissolved polypeptide, which is necessary to restore functionality. The disulfide bonds necessary for the protein to overlap properly in nature must be formed again. In prototyping procedures such as disulfide bond exchange, expensive and toxic reagents such as glutathione and oxidized 2-mercaptoethanol or dithiothritol may be used. In addition, the final yield of the bioactive protein, a genetic engineering product, may be relatively low. Moreover, even the traces of toxic substances required to dissolve and regenerate secondary and tertiary protein structures may render clinical application of the protein impossible. Thus, the production of target proteins into insoluble individuals accumulated in bacterial host cells may not only complicate the production of recombinant proteins but also make them inadequate for clinical use (Fisher, B., Sumner, I., Goodenough, P., Biotech and Bioeng. 41: 3-13, 1993).

Technical difficulties associated with the extraction of proteins produced by expressing genes of heterologous origin from bacterial host cells transformed with plasmids are directed to transforming bacterial host cells into bacteriophages whose lysis pathways lead to lysis of the host cells. It can be overcome by infection. Thus, the desired protein is simply released into the culture. (Breeze A.S., British Patent No. 2 143 238A). Therefore, Breeze disclosed a method of increasing the yield of enzymes produced in Escherichia coli by infecting bacterial cells with phage λ with temperature-sensitive cl mutations to control lysis. The cl-gene product inhibits the early stages of transcription and interferes with the transcription of the latter part of phage DNA, which is required for conjugation of the head and tail and cell lysis (Mantiatis, T., Fritsch, EF, Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory Press). Bacteriophage with temperature-sensitive cl mutations can produce and maintain a lysogenic state as long as cells multiply at a temperature that causes the cl-gene to inhibit transcription of the phage gene required for lytic growth. Thus, the transformed host cell can be cultured at 30 ° C., where transcription of phage DNA continues to be inhibited by cl and the phage remain lysed until the maximum fermentation stage is reached.

The incubation temperature is then increased to 42 ° C. for 30 minutes in order to eliminate the activity of the cl inhibitor, and the phage begin to dissolve. The host cells are then incubated at 30 ° C. for 2-3 hours to allow the enzyme to completely dissolve (Breeze A.S. British Patent No. 2 143 238A).

Breeze discloses a method of releasing proteins from bacterial producer cells, but it is necessary to incubate the producers at temperatures not exceeding 30 ° C., which is not optimal for the growth of E. coli. At temperatures above 32 ° C., synthesis at optimum temperature (37 ° C.) is not possible because the temperature sensitive solubility propagates will dissolve before reaching the maximum fermentation accumulation stage.

Furthermore, as Breeze discloses, culturing bacterial host cells at 42 ° C. for 30 minutes can activate proteolytic enzymes that degrade target proteins.

Auerbach et al. (US Pat. No. 4,637,980) used phage λ DNA fragments to induce lytic release of recombinant products. Like Breeze, such methods allow for temperature-dependent mutation of the λcl-gene product to dissolve depending on the temperature of the bacterial host cell. In Auerbach's method, the λDNA fragment retained the functions of genes N, Q, R and S encoding endorisine to produce lysozyme after inactivating the cl inhibitor at 42 ° C. Most of the remaining phage genes are removed; Mutations in the O and P genes inhibited the replication of phage DNA. As a result, the λ DNA was not a fully functioning phage capable of regulating the expression of the target gene. In addition, Auerbach's λDNA is not suitable for use as a vector carrying a target gene.

Furthermore, as described above, incubating bacterial host cells for 90-120 minutes at 42-44 ° C., as described by Auerbach, can activate proteolytic enzymes that degrade target proteins.

In addition to lysing and releasing complete proteins from bacterial host cells, bacteriophages can also be used as an alternative to bacterial plasmid-based vectors for delivering heterologous DNA to host bacterial cells (Murray, NE and Murray, K., Nature 251). : 476-481, 1974; Moir, A., Brammar, WJ, Molec. Gen. Genet. 149: 87-99, 1976). In general, amplification of genes and their products is achieved during lytic growth of phage. At this time, the genome of the phage enters the bacterial host DNA (Panasenko, SM, Cameron, JR, Davis, RV, Lehman, LR, Science 196; 188-189, 1977; Murray, NE, and Kelley, WS, Molec. Gen. Genet. 175; 77-87, 1979; Walter, F., Siegel, M., Malke, H., 1990. DD 276,694; Mory, Y., Revel, M., Chen, L., Sheldon, IF , Yuti-Chernajovsky, 1983, GB 2,103,222 A). Usually, either lysogenic cultures are used to infect bacterial host cells or "warmed" bacterial cultures in which recombinant lysogenic phage λ is used to amplify expression of genes of heterologous origin.

There have been examples of successful use of lambda vectors for the expression of genes of heterologous origin, but lambda vectors have been used primarily for gene duplication. Once cloned, the genes are transferred to plasmid vectors for more efficient expression. For example, when E. coli was infected by phage λ Charon 4A C15 containing the human β-interferon gene, the amount of interferon in the cell lysate was 7-8 × 10 ⁶ units / liter. After DNA fragments carrying the target DNA were replicated from the phage to the plasmid, the β-interferon yield was increased to 1 × 10 ⁸ units / liter (Moir, A., Brammar, WJ, Molec. Gen. Genet. 149: 87-99,1976)

In order to increase the yield of heterologous proteins produced in bacterial host cells by recombinant vectors, mutations in the phage genome have been introduced, which cause the phage λ to lose the ability to initiate bacterial cell lysis. In this way, yields were increased by prolonging the time at which a heterologous gene was expressed in bacterial host cells. Thus, for example, the yield of DNA ligase 1 in lysogenic cultures containing λgt4ligS with amber mutations in the S-gene is shown to be higher than that of DNA ligase 1 in lysogenic cultures containing λgt4lig without amber mutations. 5 times higher than the yield (Panasenko, SM, Cameron, JR, Davis, RV, Lehman, LR, Science 196; 188-189, 1977). Phage λS protein is required for dissolution; Thus, the S variant does not lyse the host cell and accumulates many intracellular phage particles as well as the target protein (Mantiatis, T., Fritsch, EF, Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory Press).

Compared to the recombinant phage lambda having no amber mutation, it has been reported that the yield of DNA polymerase 1 is similarly increased in the soluble culture containing the recombinant phage lambda having the amber mutation in the S and Q genes (Murray, NE and Kelley, WS, Molec. Gen. Genet. 175; 77-87, 1979). Phage λQ protein is required for late transcription of phage DNA, the latter comprising many genes involved in head-to-tail junctions and cell lysis (Mantiatis, T., Fritsch, EF, Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory Press).

In US Pat. No. 4,710,463, Murray discloses a method for tolerating E. coli non-inhibiting (Su °) strains with phage containing mutations in the λS and E genes as well as mutations in temperature sensitive cl. As a result, prolonged incubation of lysogenic E. coli at 37 ° C. increases recombinant protein production, which is retained intracellularly. For they are not normally lysed by the phage gene product. Since the recombinant phage genome is not wrapped in a protein membrane, it remains transcriptionable.

Although vectors with N, R, S, Q, and / or E mutations can be used to increase the yield of possible heterologous proteins, the potential technical advantages of bacteriophage vectors related to the dissolution release of the target protein are: This variation may be lost. This is because the target protein accumulates in bacterial cells. Thus, when producing a heterologous protein using a variant λ vector defective in lysis, the extraction and purification steps, as described above, for bacterial cells converted by the plasmid vector, with the loss of the resulting product, must be performed. do.

The T7 promoter / T7 RNA polymerase system is useful for expressing recombinant proteins in large quantities. To use the T7 promoter, you must have a T7 RNA polymerase. T7 RNA polymerase may be supplied by inducing recombinant T7 polymerase contained in a source in a host state or by transforming with a plasmid for expression of the T7 polymerase gene. T7 RNA polymerase is very specific to its own promoter. Transcriptional reactions from the T7 promoter are highly effective and many copies of the full length RNA can be produced from each template.

In one embodiment, a method of producing a bioactive protein is described that comprises one or more copies of an expressible gene encoding a bioactive protein linked to operate on a T7 polymerase promoter. A branched plasmid, transforming E. coli strain capable of expressing a gene of T7 RNA polymerase; Infecting the transformed bacterial host cell with a bacteriophage that can control delayed lysis; And culturing E. coli host cells until soluble and bioactive proteins are produced in desired levels.

In one preferred embodiment, the bacteriophage has a temperature sensitive variation. In a more preferred embodiment, the temperature sensitive mutation is cl ₈₅₇ .

Preferably, the E. coli host cells are grown at a temperature that prevents lytic growth of the bacteriophage before the culturing step.

In a preferred embodiment, the bacteriophage has a mutation in one or more genes that can regulate delay in lysis. In a more preferred embodiment, one or more genes capable of regulating delaying lysis are selected from the group consisting of N, Q, and R.

In a preferred embodiment, the E. coli strain produces an inhibitory factor that repairs amber variations.

In another embodiment, the E. coli strain does not have an inhibitory factor that repairs amber variations.

In a preferred embodiment, the infected bacteriophage is provided such that the multiplicity of infection is in the range of about 1-100. In a more preferred embodiment, the infected bacteriophage is provided such that the multiplicity of infection is in the range of about 10-25.

In one embodiment, the expressible gene encodes a human acid fibroblast growth factor. In another embodiment, the acidic fibroblast growth factor of human comprises 134 amino acids. In another embodiment, the acidic fibroblast growth factor of humans comprises 140 amino acids. In another embodiment, the acidic fibroblast growth factor of humans comprises 146 amino acids. In another embodiment, the human acid fibroblast growth factor comprises 155 amino acids. In the most preferred embodiment, the acidic fibroblast growth factor of human has the sequence given in SEQ ID NO: 1.

In one embodiment, the expressible gene encodes a human growth hormone. In another embodiment, the expressible gene encodes human interferon. In another embodiment, the expressible gene encodes E. coli methionine amino peptidase.

In a preferred embodiment, the gene of T7 RNA polymerase is under the control of an inducible promoter. In a more preferred embodiment, the inducible promoter is a lac UV 5 promoter.

In a preferred embodiment, there is provided a method of producing a physiologically active protein, the method comprising the steps of: a) growing a first strain of Escherichia coli living with a bacteriophage λ strain having a temperature-sensitive mutation; b) adjusting the temperature to dissolve the first strain of E. coli and release the bacteriophage? c) under the control of an inducible promoter, transformed into a plasmid having at least one copy of an expressable gene encoding a physiologically active protein operably linked to a T7 polymerase promoter, and adding an inducer to the gene of the T7 RNA polymerase. Providing a second strain of E. coli that can be induced to express; d) infecting the second strain of E. coli with the bacteriophage λ released from the first strain of E. coli; And e) the infected E. coli in a culture medium comprising an inducing factor such that the soluble, bioactive protein is produced at a concentration higher than 100 μg / ml and released into the culture medium upon dissolution of the second strain of E. coli. Culturing the second week.

The invention disclosed herein also includes chemically synthesized nucleic acids consisting essentially of the sequence given in SEQ ID NO: 1.

To summarize the present invention and its advantages over the prior art, the objects and advantages of the present invention have been described above. Of course, it should be understood that not all of the objects and advantages may necessarily be achieved in accordance with any embodiment of the present invention. Thus, for example, those skilled in the art will appreciate that the invention may be practiced in a manner that achieves or optimizes one or a group of advantages without necessarily achieving the other objects and advantages as disclosed or suggested herein. You will know.

Other aspects, features, and advantages of the present invention will become apparent from the detailed description of the embodiments described below.

These and other features of the invention are described with reference to the drawings of the embodiments, which are presented for purposes of illustration only and are not intended to limit the scope of the invention.                 

1 is a chemically synthesized nucleotide sequence of human acidic fibroblast growth factor (155 amino acids) (SEQ ID NO: 1), which replaces a naturally occurring codon with a codon found in a high level expressed E. coli protein. It is also a diagram showing the amino acid sequence (SEQ ID NO: 2) translated therefrom.

Figure 2 shows the changes in chemically synthesized haFGF 155 codon, FGF fr HUMECGFB is a sequence obtained from the NCBI gene bank (SEQ ID NO: 3). HaFGF 155 shows a chemically synthesized sequence (SEQ ID NO: 1) according to one embodiment of the present invention.

FIG. 3 shows pET24-155 @ rev construct comprising a chemically synthesized haFGF 155 gene (SEQ ID NO: 1).

Figure 4 shows haFGF 155 purified by HPLC, in this electrophoretic plot, lane 1 is a 10 L controlled culture containing preparative haFGF 155, lane 2 is haFGF 155 purified with heparin-sepharose (0.45 g). / l) 7L, lane 3 corresponds to 14L extracted from 80L haFGF 155 purified by HPLC.

FIG. 5 shows pET24-134 @ rev construct comprising a chemically synthesized haFGF 134 gene (SEQ ID NO: 4). FIG.

FIG. 6 is a chemically synthesized nucleotide sequence of human acidic fibroblast growth factor (134 amino acids) (SEQ ID NO: 4), in which naturally occurring codons are substituted with codons found in high expressed E. coli protein It is also a diagram showing the amino acid sequence (SEQ ID NO: 5) translated therefrom.                 

FIG. 7 depicts pET24-140 @ rev construct comprising a chemically synthesized haFGF 140 gene (SEQ ID NO: 6). FIG.

FIG. 8 is a chemically synthesized nucleotide sequence of human acid fibroblast growth factor (140 amino acids) (SEQ ID NO: 6), which replaces a naturally occurring codon with a codon found in a high level expressed E. coli protein. And amino acid sequence (SEQ ID NO: 7) translated therefrom.

FIG. 9 shows a pET24ap-inf @ rev construct comprising a chemically synthesized interferon-2b gene (SEQ ID NO: 10).

FIG. 10 is a chemically synthesized nucleotide sequence of human interferon-2b (SEQ ID NO: 10), in which a naturally occurring codon is substituted with a codon found in a high level expressed E. coli protein and also translated therefrom. Is a diagram showing the amino acid sequence (SEQ ID NO: 11).

FIG. 11 depicts a 12.5% SDS polyacrylamide gel containing protein produced by the method using the phage described herein, where lanes are molecular weight standards containing 2 g of each standard solution; Lane 2 is 40 liters of culture containing recombinant FGF 134 protein; Lane 3 is 40 L of culture medium containing recombinant FGF 140 protein; Lane 4 was 40 liters of culture containing recombinant interferon 2B; Lane 5 is 40 L of culture medium containing recombinant FGF 155 protein; Lane 6 is 40 L of culture medium containing recombinant human growth hormone; Lane 7 was 40 liters of culture containing recombinant methionine aminopeptidase; Eight lanes are diagrams showing that the culture solution containing galactosidase of E. coli 40 L.                 

FIG. 12 depicts a 12.5% SDS polyacrylamide gel comprising recombinant protein purified according to the invention claimed herein, wherein lane 1 is the molecular weight standard; Lane 2 is 5 g of purified FGF 134 protein; Lane 3 is 5 g of purified FGF 140 protein; Lane 4 is 5 g of purified FGF 146 protein; Lane 5 is 5 g of purified interferon 2B; Lane 6 is 5 g of purified FGF 155 protein; Lane 7 is 5 g of purified methionine amino peptidase protein; 8 lane shows the molecular weight standard.

While the described embodiments represent preferred embodiments of the invention, those skilled in the art should understand that changes may be made without departing from the spirit of the invention. Accordingly, the scope of the invention should only be determined by the appended claims.

Bacteriophage λ is useful as a vector because more than 40% of the viral genome is not essential for lytic growth. This part of the lambda genome, located in the middle of the lambda DNA between genes J and N, can be replaced with heterologous DNA encoding the desired product. This part is transcribed early in the infection.

In order to maximize the expression of the target gene whose synthesis information is recorded in the region of the initial gene of the phage, special phage development conditions must be provided to ensure proper replication.

Moreover, transcription of the initial region containing the target gene should be encouraged, while transcription of late genes involved in cell lysis should be inhibited. This delays the maturation of X particles and subsequent cell lysis. As a result, early phage products, including target gene products, will accumulate in bacterial cells. Inhibition of late transcription and thereby delayed expression of the target gene may include: (1) mutation of the phage genome that inhibits late gene expression, (2) increased multiplicity of infection, and / or (3) infection at reduced temperatures. It can be achieved through the culture of bacterial cells.

The advantage of infecting producer cells with bacteriophage is that the phage allows for deep rearrangement of all macromolecular synthesis in bacterial host cells. By stopping the transcription of bacterial genes, phage can increase replication of the target gene, resulting in more desired product.

In one embodiment of the mass production method of the present invention, phage λ with amber variation (eg, Q and R-mutation) that delays bacterial dissolution is provided in an E. coli strain, denoted by Su °, wherein It does not have inhibitory factors that modify amber mutations. In order to obtain an uninhibited (Su °) strain of E. coli, the Su ° clone is a wild species Su + It is selected from the group. Preferably, a selection marker, such as resistant tetracycline or ampicillin, is inserted into phage DNA. Selection of the E. coli non-suppressive (Su °) strain, eg, E. coli K802, was performed with phage λcl ₈₅₇ Nam7Nam53 bla tet (hereinafter referred to as λbla N '). E. coli strain C600 (λbla N ') served as a source of phage. This phage was obtained by inserting the plasmid pCV11 (bla tet) at the EcoRI site into a single site (EcoRI) vector with ts-mutation in the inhibitory gene (cl ₈₅₇ ). Two amber mutations were then introduced into the phage N gene by in vivo recombination.

The clones were cost-tested with phage λ removed (phage clear). In addition to phage λbla N ′, phage λcl ₈₅₇ Q _am117 R _am54 was used for testing inhibitory factors.

As is known, phage λN ′ variants are unable to lyse host cells and exist intracellularly in the form of highly unstable plasmids. If the host cell contains inhibitory factors, the amber mutation is modified according to the phenotype, the N protein is synthesized, and the phage can be lysed and grown. Su + This difference in viability of cells and Su 'cells infected by N' is used as the cornerstone for selecting the Su 'return mutations that spontaneously emerge from the E. coli Su ⁺ cell population. Phage, along with the inserted plasmid, introducing resistant ampicillin and tetracillin into the cells, were used to prevent unlying Su ° cells from interfering with the variant search. The phage also includes ts-variants of inhibitory factors that allow lytic growth of the phage resulting in cell lysis.

The medium containing ampicillin and tetracycline was infected with phage λbla N 'and then grown at 43 ° C. and then inoculated with Su ⁺ culture, without any single inhibitory factor containing phage λbla N' in the form of a plasmid. The cells will form on the plates. The cells are healed from the phage to obtain a Su ° derivative of the parent culture. This method can be broken down into several steps:

1. Infection of culture with phage λbla N '

E. coli Su ⁺ cultures were grown in M9 medium with maltose at 37 ° C. with vigorous shaking at a concentration of 1 × 2 × 10 ⁸ cells / ml. The cells were infected with phage λbla N ′ at 5-10 multiplicity per cell and incubated at 20 ° C. for 20 minutes. Under given conditions, the infection efficiency is about 100%, and in addition to the Su ⁺ cell stock, the phage also infects one Su ° cells.

2. Selecting Cells Without Inhibitors Including Marker Phage

After infection, cells were planted in agar medium with 12γ / ml tetracycline and 20γ / ml ampicillin and grown at 43 ° C. After 24 hours, one colony was formed, which was transferred to Agar medium with antibiotics and planted at 37 ° C.

3. Healing clones selected from phage λbla N '

Since phage λ N ′ in E. coli Su ° cells are in the form of highly unstable plasmids, they were planted in selected agar medium without antibiotics and grown at 37 ° C. to cure clones selected from these phages. The number of cells that lost the phage once through the medium without antibiotics reached 12-35%.

These cells were selected by investigating loss of antibiotic resistance and gaining sensitivity to phage λ clear.

4. Testing for Inhibitors in the Cell

Phage λ with amber-mutation was tested for the ability to form plagues on the healed clones.

Homologous derivatives without parental Escherichia coli Su ⁺ strains were clones, phage λbla N 'on the clone did not form a villain, and phage λcl ₈₅₇ Q _am117 R _am54 produced 1-3 _P10 ⁵ PFU / mL Phage cl ₈₅₇ without mutation in genes Q and R produced 1 × 10 ¹⁰ PFU / mL.

Using this method, we obtained a Su ° return mutant of Escherichia coli K802 Su ⁺ . Based on the number of cells at the time of infection and the number of Su ° return mutants therein, the cycle in which cells without inhibitory factors appeared was 3 × 10 ⁻⁷ .

In a preferred embodiment, the gene of interest is replicated into pET-24a (+) under the control of the T7 promoter. Any gene of interest can be used to practice the claimed invention. Specific examples include but are not limited to human growth hormone, interferon, methionine amino peptidase, human aFGF 134 amino acid form, human aFGF 140 amino acid form, human aFGF 146 amino acid form, and human aFGF 155 form. Do not. In other embodiments, the gene of interest can be replicated into bacterial plasmids and phage under the control of an appropriate promoter. In the most preferred embodiment, the chemically synthesized haFGF 155 gene (SEQ ID NO: 1) is replicated into pET-24a (+) under the control of the T7 promoter. The T7 promoter is only recognized by T7 RNA polymerase and not by E. coli RNA polymerase. Plasmids obtained with the haFGF 155 gene (phaFGF 155) are transformed into E. coli BL21 (DE3). This state contains the t7 RNA polymerase gene. The T7 RNA polymerase gene is under the control of an inducible lac UV5 promoter to induce the synthesis of T7 RNA polymerase only when necessary. Because the protein is toxic to E. coli cells. Induction of the lac promoter is performed by adding IPTG to the nutrient medium. In order to obtain the haFGF 155 protein, a producer strain comprising a recombinant plasmid together with the haFGF 155 gene was subjected to strong aeration at a cell density of 5 x 10 ⁷ to 5 x 10 ⁹ cells / ml at a temperature of 20 to 40 ° C. Incubated under The producer line is then infected with lambda phage with a ts-variable cl inhibitory factor gene at a multiplicity of 0.1 to 100 phages per cell, and the culture is continued for 2 to 14 hours at 20 to 37 ° C. Along with the phage, 1 mM of IPTG is also introduced.

The haFGF gene encodes a gene comprising 155 amino acid residues. However, it was possible to separate only two short aFGF forms from tissue samples. The two separate forms include 140 and 134 amino acid residues. The aFGF form comprising 140 amino acids is considered complete, whereas the aFGF form comprising 134 amino acids is considered cleaved. It was not possible to extract aFGF forms comprising 155 amino acids from tissue samples. It is not known whether the short isoform appears as a normal function in cell processing or is an artificial product produced during isolation by a specific protease during aFGF extraction.

Western blot analysis of the proteins produced from the DNA recombinant molecules isolated for the three aFGF forms showed that the 140 and 134 forms were expressed more and the 155 forms expressed less.

In a preferred embodiment of the invention, the gene of human acidic fibroblast growth factor encodes the 144 amino acid form of the aFGF protein and is chemically synthesized (SEQ ID NO: 1). The nucleotide sequence of the haFGF 155 gene was inferred based on the previously described haFGF amino acid sequence (SEQ ID NO: 2). The amino acid sequence of the synthesized haFGF gene is not different from the previously described sequences, such as the translated sequence of the FGF nucleotide sequence of SEQ ID NO: 3. However, the preferred nucleotide sequence of haFGF is different from the sequences already described. In a preferred embodiment of the present invention, the haFGF 155 gene was chemically synthesized using codons frequently used by Escherichia coli for bacterial protein synthesis. Codon usage tables for E. coli are well known and available.

See, for example, http://psyche.uthct.edu/shaun/Sblack/codonuse.html . Chemical synthesis of human aFGF genes was performed by well known methods (Edge et al. (1983) Nucleic Acids Research 11 (18): 6419-6435).

In contrast, any gene of interest may comprise animal tissues encoding other forms of haFGF known to those of skill in the art, including the 146, 140, and 134 homologous forms and variants, derivatives, analogs or fragments thereof. It can be used to practice the present invention, including but not limited to DNA isolated from. Genes encoding human growth hormone, human interferon, and E. coli methionine amino peptidase are also examples of these.

Figure 1 shows the complete nucleotide sequence of the haFGF 155 gene as synthesized by the inventors (SEQ ID NO: 1) and the sequence of human acid fibroblast growth factor (SEQ ID NO: 3) obtained from the gene bank. give.

These two sequences are compared in FIG. 2, and 80 codons are distinguished.

Expression and clone vectors generally have a promoter recognized by the host organism and are operably linked to the gene of interest. Promoters are untranslated sequences located above (5 ') the start codon of structural genes (typically within 100 to 1,000 base pairs) that regulate the transcription and translation of the specific nucleic acid sequences to which they are linked. Such promoters are generally divided into two classes, inducible and structural. Inducible promoters are promoters that begin to increase the level of transcription from DNA under the control of these promoters in response to changes in culture conditions, eg, the presence or absence of certain nutrients or changes in temperature. Many promoters are now known which are recognized by prokaryotic host cells. Those skilled in the art will know how to link them to the gene of interest using appropriate linkers or auxiliaries to provide appropriate cleavage sites.                 

Transformation means introducing DNA into an organism, and the introduced DNA can be replicated by assimilating into chromosomes or as elements outside the chromosomes. Transformation of prokaryotic cells is carried out using CaCl ₂ or using techniques well known to those skilled in the art, such as electroporation. Producer strains were cultured under conditions that delayed the lysed growth of lambda phage to produce large amounts of recombinant protein. Such conditions include a drop in culture temperature and the use of amber variations of late lambda phage such as the Q and R genes.

Recombinant protein accumulates in the culture medium as a protein that can be lysed as a result of lysis of the producer primary cells by lambda phage. The yield of recombinant protein amounts to 20% of the soluble protein accumulated in the culture medium. Debris is centrifuged and removed from the culture medium.

The recombinant protein can then be purified from the soluble soluble proteins and peptides according to purification methods well known to those skilled in the art. Such methods include, but are not limited to, separation on ion exchange columns, ethanol precipitation, reverse phase HPLC, immunoaffinity, SDS-PAGE, ammonium sulfate precipitation, and gel filtration procedures. For haFGF protein, haFGF protein can be applied to heparin sepharose to obtain purified haFGF.

A more detailed description of the invention is provided below. While the described embodiments represent preferred embodiments of the invention, it should be understood that changes may be made by those skilled in the art without departing from the spirit of the invention. Accordingly, the scope of the invention should be determined entirely by the appended claims.

<Example 1>

Production of human aFGF 155 by using phage

Cultures of Escherichia coli BL21 (DE3) (NOVAGEN) were transformed by plasmid pET24 155 @ rev (FIG. 3). This plasmid contains a copy of the haFGF 155 gene, which encodes the human acid fibroblast growth factor (155 amino acids).

BL21 (DE3) cultures contain a copy of the gene for T7 RNA polymerase under the control of an inducible lac UV5 promoter in the bacterial genome (Studier et al. (1986) J. Mol. Biol. 189: 113-130 ). To produce plasmid pET24-155 @ rev, a chemically synthesized haFGF 155 gene (SEQ ID NO: 1) was inserted into plasmid pET-24a (+) (NOVAGEN) under the control of the T7 promoter. The expression of the haFGF 155 gene is initiated only after the T7 polymerase in mediated cells is revealed through the induction of lac UV5 promoter by IPTG.

The culture of Escherichia coli BL21 (DE3) with pET24-155 @ rev was grown with stirring at 37 ° C. at a density of 2 × 10 ⁸ cells / ml in LB medium containing 50 g / ml kanamycin. The cells were then infected with phage cl ₈₅₇ Q _am117 R _am54 at a _multiplicity of about 10 phages per bacterial cell and incubated with stirring at 21 ° C. for about 14 hours. With phage, 1 mM of IPTG was also introduced into the medium.

Phage cl ₈₅₇ Q _am117 R _am54 was prepared from lysogenic _cultures of Escherichia coli RLMI, and the cultures were grown to a density of about 1 × 10 ⁵ cells / ml with strong aeration in LB medium at 30 ° C. The lysogenic cultures were warmed at 43 ° C. and incubated for 20 minutes to inactivate the cl inhibitory factor. The temperature was then lowered to 37 ° C., and after 60-70 minutes the bacterial cells were lysed with phage forming at 1-2 × 10 ¹⁰ PFU / ml.

After culturing the cells infected with the phage for 14 hours, the debris was centrifuged to remove from the culture. Cultures containing haFGF 155 protein were applied to a heparin Sepharose column to obtain pure haFGF 155.

Cultures containing haFGF 155 were analyzed by SDS-polyacrylamide gel under staining conditions and stained with Coomassie Blue. In FIG. 4, electrophoretic photographs containing haFGF protein were compared with purified haFGF protein. Lane 1 is for 10 μl of the culture medium, Lane 2 is for 7 μl of haFGF 155 protein (0.45 g / l) purified with heparin-sepharose, and Lane 3 is for 80 μl of FGF-155 purified by HPLC. And the left unmarked lanes are for the molecular weight standard (Amersham Pharmacia Biotech). The production of haFGF 155 protein in phage-infected cultures was about 20% of the total cellular protein.

The molecular weight of haFGF was 17,908 Daltons as measured by Densitometer Image Master VDS (data not shown).

Human aFGF 155 produced according to the method disclosed above had physiological activity based on chick cell membrane assay (Example 6). In addition, purified human aFGF 155 showed physiological activity in proliferation assays based on cells using BALB / c 3T3 fibroblasts (Linemeyer, US Pat. No. 6,640,32). Half the maximum cell proliferation was when the concentration of aFGF 155 was 32 pg / ml. Unpurified human aFGF 155 in bacterial culture also showed the same physiological activity as purified aFGF 155 in the 3T3 fibroblast assay. This indicates that aFGF 155 was initially synthesized in bacteria as a soluble and bioactive protein.

<Example 2>

Production of human aFGF 134 amino acid form by phage method

Culture of Escherichia coli BL21 (DE3) (NOVAGEN) is transformed by plasmid pET24 134 @ rev (FIG. 5), which encodes human aFGF (134 amino acids) (FIG. 6; SEQ ID NO: 4). One copy of the chemically synthesized gene is included. The translated amino acid sequence is shown in SEQ ID NO: 5. BL21 (DE3) cultures contain a copy of the gene for T7 RNA polymerase under the control of an inducible lac UV5 promoter in the bacterial genome (Studier et al. (1986) J. Mol. Biol. 189: 113-130 ). A gene in human aFGF 134 amino acid form was inserted into plasmid pET-24a (+) (NOVAGEN) under the control of the T7 promoter. Expression of the aFGF 134 gene is initiated only after T7 polymerase in mediated cells is revealed through the induction of lac UV5 promoter by IPTG.

Cultures of E. coli BL21 (DE3) with plasmid pET24-134 @ rev were grown with stirring at 37 ° C. at a density of 2 × 10 ³ cells / ml in LB medium containing 50 g / ml kanamycin. The cells were then infected with phage cl ₈₅₇ Q _am117 R _am54 at a _multiplicity of about 10 phages per bacterial cell and incubated with stirring at 21 ° C. for about 14 hours. With phage, 1 mM of IPTG was also introduced into the medium.

Phage cl ₈₅₇ Q _am117 R _am54 was prepared from lysogenic _cultures of E. coli RLMI, and the cultures were grown to a density of about 1 × 10 ⁸ cells / ml with strong aeration in LB medium at 30 ° C. The lysogenic cultures were warmed at 43 ° C. and incubated for 20 minutes to inactivate the cl inhibitory factor. The temperature was then lowered to 37 ° C., and after 60-70 minutes the bacterial cells were lysed with phage forming at 1-2 × 10 ¹⁰ PFU / ml.

After culturing the cells infected with the phage for 14 hours, the debris was centrifuged to remove from the culture. Cultures containing haFGF 155 protein were applied to a heparin Sepharose column to obtain pure haFGF 155.

<Example 3>

Production of human aFGF 134 amino acid form by phage method

Culture of E. coli BL21 (DE3) (NOVAGEN) is transformed by plasmid pET24 140 @ rev (Fig. 7), which encodes human aFGF (Fig. 8; 140 amino acids) (SEQ ID NO: 4). One copy of the chemically synthesized gene is included. The protein is shown in SEQ ID NO: 7. BL21 (DE3) cultures contain a copy of the gene for T7 RNA polymerase under the control of an inducible lac UV5 promoter in the bacterial genome (Studier et al. (1986) J. Mol. Biol. 189: 113-130 ). A gene in human aFGF 140 amino acid form was inserted into plasmid pET-24a (+) (NOVAGEN) under the control of the T7 promoter. Expression of the aFGF 140 gene is initiated only after the T7 polymerase in the mediated cells is expressed through the induction of the lac UV5 promoter by IPTG.

Cultures of Escherichia coli BL21 (DE3) with pET24-140 @ rev were grown with stirring at 37 ° C. at a density of 2 × 10 ⁸ cells / ml in LB medium containing 50 g / ml kanamycin. The cells were then infected with phage cl ₈₅₇ Q _am117 R _am54 at a _multiplicity of about 10 phages per bacterial cell and incubated at 21 to about 14 hours with stirring. With phage, 1 mM of IPTG was also introduced into the medium.

Phage cl ₈₅₇ Q _am117 R _am54 was prepared from lysogenic _cultures of E. coli RLMI, and the cultures were grown to a density of about 1 × 10 ⁸ cells / ml with strong aeration in LB medium at 30 ° C. The lysogenic cultures were warmed at 43 ° C. and incubated for 20 minutes to inactivate the cl inhibitory factor. Thereafter, the temperature was lowered to 37 ° C., and after 60 to 70 minutes, the bacterial cells were lysed with phage forming at 1-2 × 10 ¹⁰ PFU / ml.

After culturing the cells infected with the phage for 14 hours, the debris was centrifuged to remove from the culture. Cultures containing haFGF 140 protein were applied to a heparin Sepharose column to obtain pure haFGF 140.

Human aFGF 140 produced according to the method had physiological activity based on chick cell membrane assay (Example 6).

<Example 4>

Production of human aFGF 146 amino acid form by using phage

Cultures of Escherichia coli BL21 (DE3) (NOVAGEN) were transformed by plasmid pET24-146 @ rev (FIG. 7), which was chemically synthesized encoding human aFGF (146 amino acids) (not shown). Contains one copy of the gene. BL21 (DE3) cultures contain a copy of the gene for T7 RNA polymerase under the control of an inducible lac UV5 promoter in the bacterial genome (Studier et al. (1986) J. Mol. Biol. 189: 113-130 ). A gene in human aFGF 146 amino acid form was inserted into plasmid pET-24a (+) (NOVAGEN) under the control of the T7 promoter. Expression of the aFGF 146 gene is initiated only after T7 polymerase in mediated cells is revealed through the induction of lac UV5 promoter by IPTG.

Cultures of Escherichia coli BL21 (DE3) with pET24-146 @ rev were grown with stirring at 37 ° C. at a density of 2 × 10 ⁵ cells / ml in LB medium containing 50 g / ml kanamycin. The cells were then infected with phage cl ₈₅₇ Q _am117 R _am54 at a _multiplicity of about 10 phages per bacterial cell and incubated with stirring at 21 ° C. for about 14 hours. With phage, 1 mM of IPTG was also introduced into the medium.

Phage cl ₈₅₇ Q _am117 R _am54 was prepared from lysogenic _cultures of E. coli RLMI, and the cultures were grown to a density of about 1 × 10 ⁸ cells / ml with strong aeration in LB medium at 30 ° C. The lysogenic cultures were warmed at 43 ° C. and incubated for 20 minutes to inactivate the cl inhibitory factor. Thereafter, the temperature was lowered to 37 ° C., and after 60 to 70 minutes, the bacterial cells were lysed with phage forming at 1-2 × 10 ¹⁰ PFU / ml.

After culturing the cells infected with the phage for 14 hours, the debris was centrifuged to remove from the culture. A culture containing haFGF 146 protein was applied to a heparin sepharose column to obtain pure haFGF 146.

Human aFGF 146 produced according to the method had physiological activity based on chick cell membrane assay (Example 6).

<Example 5>

Purification of Recombinant haFGF Protein

Cultures containing haFGF protein were diluted once with 0.04M KH ₂ PO ₄ buffer at pH 7.0 and applied to a Heparin-Sepharose column equilibrated with 0.02M KH ₂ PO _{4 at} pH 7.0. The flow rate was adjusted to 80 ml / hour. After the culture solution containing the haFGF protein was applied to the column, the column was washed with 0.02M KH ₂ PO ₄ buffer, pH 7.0. Next, the column was washed with 0.02M KH ₂ PO ₄ buffer containing 0.6M NaCl at pH 7.3.

Elution was performed using 0.02 M KH ₂ PO ₄ buffer with 1.5 M NaCl at pH 7.5. All steps were performed at 4.

<Example 6>

A study on the effect of FGF on the formation of new blood vessels in the CAM of chicken embryos

Angiogenesis method for chicken embryo model (Thomas et al. (1985) Proc. Natl. Acad. Sci., USA 82: 6409-6413) was employed to compare to pure brain-derived acidic fibroblast growth factor The effect of haFGF 155, 146, 140 recombinant proteins on angiogenesis was measured. Pure brain-derived acidic fibroblast growth factor is a potential angiogenic endothelial cell mitogen (dividing promoter) with sequence homology with interleukin.

The skin of 3 day old chicken embryos was sterilized with ethyl alcohol. The shell and the undershell were removed from the air box using forceps and the eggs were covered with a 35 mm plastic Petri dish. The embryos were incubated at 37 ° C. for 5-6 days. After this period, embryos were examined to select eggs with well-developed vessels of CAM for experimentation.

Disc-shaped filter paper deposited with a gel containing FGF was placed on egg CAM with the gel facing the vessel and incubated in a thermostat at 37 ° C. for 3 more days. The gel was prepared as follows: FGF tested amount was dissolved in 30 liters of Eagle's solution (solution 1); Thereafter, 10 g of heparin was dissolved in 30 liters of the eagle solution and 2% agarose was added (solution 2). Thereafter, the same volumes of solutions 1 and 2 were mixed and the resulting mixture was deposited on a disc filter paper of 12 mm diameter in a quantity of 60 liters.

On day 4, the filter paper was removed. Less than about 1 ml thick cow milk (10% milk fat) was injected under CAM. As a result, CAM vessels were easily observed under a white background.

The results of the experiment were recorded in a video camera in conjunction with a computer. The formation of new CAM vessels under the influence of FGF was assessed by the following coefficients: the nature and direction of vessel growth, their quantity and quality (large, medium, small), the presence and absence of junctions, etc. These data were compared with samples from controls not exposed to FGF. On day 14 of growth chicken embryo blood vessels were treated with FGF 155 produced according to the recombinant method using the phage described herein and purified with heparin sepharose as described.

Recombinant FGF 155 protein was used to demonstrate the formation of new blood vessels. On day 4 after using 1 g of FGF 155, the blood vessels were primarily small in size and grew radially. Increasing the amount of FGF 155 to 3g, the size of the blood vessels was seen to increase. Medium blood vessels were observed to grow radially. When FGF 155 was further increased to 4 g, the blood vessels of the large, medium and small cows appeared on the 4th day compared to the control group.

<Example 7>

Production of human growth hormone by the method of using phage

Cultures of Escherichia coli BL21 (DE3) (NOVAGEN) were transformed with a plasmid containing a copy of a chemically synthesized gene (SEQ ID NO: 8) encoding human growth hormone. The translated amino acid sequence is shown in SEQ ID NO: 9. BL21 (DE3) cultures contain a copy of the gene for T7 RNA polymerase under the control of an inducible lac UV5 promoter in the bacterial genome (Studier et al. (1986) J. Mol. Biol. 189: 113-130 ). Human growth hormone genes were inserted into plasmid pET-24a (+) (NOVAGEN) under the control of the T7 promoter. Expression of the aFGF 146 gene is initiated only after T7 polymerase in mediated cells is revealed through the induction of lac UV5 promoter by IPTG.

The transformed culture of Escherichia coli BL21 (DE3) was grown with stirring at 37 ° C. at a density of 2 × 10 ⁸ cells / ml in LB medium containing 50 g / ml kanamycin. The cells were then infected with phage cl ₈₅₇ Q _am117 R _am54 at a _multiplicity of about 10 phages per bacterial cell and incubated with stirring at 21 ° C. for about 14 hours. With phage, 1 mM of IPTG was also introduced into the medium.

Phage cl ₈₅₇ Q _am117 R _am54 was prepared from lysogenic _cultures of E. coli RLMI, and the cultures were grown to a density of about 1 × 10 ⁸ cells / ml with strong aeration in LB medium at 30 ° C. The lysogenic cultures were warmed at 43 ° C. and incubated for 20 minutes to inactivate the cl inhibitory factor. The temperature was then lowered to 37 ° C., and after 60-70 minutes the bacterial cells were lysed with phage forming at 1-2 × 10 ¹⁰ PFU / ml.

After culturing the cells infected with the phage for 14 hours, the debris was centrifuged to remove from the culture. Human growth hormone proteins were purified by column chromatography in a manner known to those skilled in the art to obtain pure human growth hormone. As a result of analysis by cell-based bioassay using Nb2 lymphoma cells (Gout PW, Cancer Research 40: 2433-2436, 1980), the purified human growth hormone had physiological activity. The concentration of human growth hormone giving half of Nb2 cell growth stimulation was 125 pg / ml.

<Example 8>

Production of Human Interferon-2b by Using Phage

Cultures of Escherichia coli BL21 (DE3) (NOVAGEN) were transformed with plasmid pET24ap-inf @ rev (FIG. 9). This plasmid contains a copy of a chemically synthesized gene encoding FIG. 2 human interferon (FIG. 10; SEQ ID NO: 10). The translated amino acid sequence is shown in SEQ ID NO: 11. BL21 (DE3) cultures contain a copy of the gene for T7 RNA polymerase under the control of an inducible lac UV5 promoter in the bacterial genome (Studier et al. (1986) J. Mol. Biol. 189: 113-130 ). The interferon gene was inserted into plasmid pET-24a (+) (NOVAGEN) under the control of the T7 promoter. Expression of the interferon gene is initiated only after T7 polymerase in mediated cells is shown through the induction of lac UV5 promoter by IPTG.                 

The culture of Escherichia coli BL21 (DE3) together with the plasmid pET24ap-inf @ rev was grown with stirring at 37 ° C. at a density of 2 × 10 ⁸ cells / ml in LB medium containing 50 g / ml kanamycin. The cells were then infected with phage cl ₈₅₇ Q _am117 R _am54 at a _multiplicity of about 10 phages per bacterial cell and incubated with stirring at 21 ° C. for about 14 hours. With phage, 1 mM of IPTG was also introduced into the medium.

Phage cl ₈₅₇ Q _am117 R _am54 was prepared from lysogenic _cultures of E. coli RLMI, and the cultures were grown to a density of about 1 × 10 ⁸ cells / ml with strong aeration in LB medium at 30 ° C. The lysogenic cultures were warmed at 43 ° C. and incubated for 20 minutes to inactivate the cl inhibitory factor. The temperature was then lowered to 37 ° C., and after 60-70 minutes the bacterial cells were lysed with phage forming at 1-2 × 10 ¹⁰ PFU / ml.

After incubating the phage infected cells for 14 hours, the debris was centrifuged and removed from the culture. Interferon was purified by column chromatography in a manner known to those skilled in the art to obtain pure interferon.

Based on the interferon antiviral assay (Aebersold P, Methods in Enzymology 119: 579-592, 1986) performed on bovine kidney cells infected with vesicular stomatitis virus, the interferon produced according to the method disclosed herein exhibited physiological activity. I had. Interferon alpha 2b had a biological potency of 1.81 × 10 ⁸ IU per mg of protein in this assay. Interferon alpha 2b contained in bacterial cultures prior to purification had the same efficacy as purified interferon in this antiviral assay. This indicates that interferon alpha 2b is initially synthesized as a protein soluble and bioactive in bacteria.

Example 9

Production of Escherichia Coli Methionine Amino Peptidase According to Method Using Phage

Cultures of E. coli BL21 (DE3) (NOVAGEN) were transformed with a plasmid containing one copy of a chemically synthesized gene encoding E. coli methionine amino peptidase. BL21 (DE3) cultures contain a copy of the gene for T7 RNA polymerase under the control of an inducible lac UV5 promoter in the bacterial genome (Studier et al. (1986) J. Mol. Biol. 189: 113-130 ). The E. coli methionine amino peptidase gene was inserted into plasmid pET-24a (+) (NOVAGEN) under the control of the T7 promoter. The expression of the E. coli methionine amino peptidase gene is initiated only after T7 polymerase in mediated cells is revealed through the induction of the lac UV5 promoter by IPTG.

The transformed culture of Escherichia coli BL21 (DE3) was grown with stirring at 37 ° C. at a density of 2 × 10 ⁸ cells / ml in LB medium containing 50 g / ml kanamycin. The cells were then infected with phage cl ₈₅₇ Q _am117 R _am54 at a _multiplicity of about 10 phages per bacterial cell and incubated with stirring at 21 ° C. for about 14 hours. With phage, 1 mM IPTG was also introduced into the medium.

Phage cl ₈₅₇ Q _am117 R _am54 was prepared from lysogenic _cultures of E. coli RLMI, and the cultures were grown to a density of about 1 × 10 ⁸ cells / ml with strong aeration in LB medium at 30 ° C. The lysogenic cultures were warmed at 43 ° C. and incubated for 20 minutes to inactivate the cl inhibitory factor. Thereafter, the temperature was lowered to 37 ° C., and after 60 to 70 minutes, the bacterial cells were lysed with phage forming at 1-2 × 10 ¹⁰ PFU / ml.

After culturing the cells infected with the phage for 14 hours, the debris was centrifuged to remove from the culture. E. coli methionine amino peptidase was purified by column chromatography in a manner known to those skilled in the art to obtain pure E. coli methionine amino peptidase.

<Example 10>

Gel analysis of recombinant protein produced according to the method using phage

SDS-polyacrylamide gel electrolysis under conditions that denature a culture comprising human aFGF 134 amino acid form, human aFGF 140 amino acid form, human aFGF 155 amino acid form, human growth hormone, interferon, and methionine aminopeptidase Analysis was carried out by electrophoresis and stained with Coomassie blue. An electrophoretic plot of a culture comprising human aFGF 134 amino acid form, human aFGF 140 amino acid form, human aFGF 146 amino acid form, human growth hormone, and interferon proteins is compared to the molecular weight standard in FIG. 11. Lane 2 is for 30 liters of culture containing human aFGF 134 amino acid form. Lane 3 is for a 30 L culture medium containing human aFGF 140 protein. Lane 4 is for 30 L of culture medium containing recombinant interferon. Lane 5 is for 30 liters of culture containing recombinant FGF 155 protein. Lane 6 is for 30 L of culture medium containing recombinant human growth hormone. Lane 7 is for 30 liters of culture containing recombinant methionine amino peptidase. Lane 1 is for 2 g of each molecular weight standard (Amersham Pharmacia Biotech). From above, the molecular weight standard is: 94,000; 67,000; 43,000; 30,000; 20,100; And 14,400.

Mixed human aFGF 134 amino acid form, human aFGF 140 amino acid form, human aFGF 155 amino acid form, human growth hormone, interferon, and methionine aminopeptidase are used to measure colored protein bands on polyacrylamide gels. Quantification was performed by scanning with Image Master VDS (Pharmacia Biotech). The production of recombinant protein in phage-infected cultures was about 20% of the total cellular protein.

Electrophoretic plots comprising recombinant haFGF 134, haFGF 140, haFGF 146, interferon, haFGF 155 and methionine aminopeptidase were compared to molecular weight standards (FIG. 12). Lane 2 is for 5 g of purified aFGF 134 protein. Lane 3 is for 5 g of purified human aFGF 140. Lane 4 is for 5 g of purified human aFGF 146 amino acid form. Production of human aFGF amino acid forms in phage-infected cultures was about 20% of the total cellular protein. Lane 5 is for 5 g of purified interferon. Lane 6 is for 5g of haFGF 155 protein. Lane 7 is for 5 g of purified E. coli methionine amino peptidase. Lanes 1 and 8 are for 2 g of molecular weight standard (Amersham Pharmacia Biotech), respectively.

Those skilled in the art will appreciate that various modifications are possible without departing from the spirit of the invention. Therefore, it is to be understood that the embodiments of the present invention are illustrative only and are not intended to limit the scope of the present invention.

<110> Phage Biotechnology Corporation <120> PHAGE-DEPENDENT SUPER PRODUCTION OF BIOLOGICALLY ACTIVE PROTEIN          AND PEPTIDES <130> PHAGE.006VPC <150> 09 / 318,288 <151> 1999-05-25 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 630 <212> DNA <213> Artificial Sequence <220> <223> This sequence was chemically synthesized based upon the amino          acid sequence of human acidic fibroblast growth factor (155 amino          acids) using codons which are used in highly expressed proteins          from E. coli. <221> CDS (222) (122) .. (586) <400> 1 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt 60 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca 120 t atg gct gaa ggg gaa atc acc acc ttt aca gcg tta acg gag 163            Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu              1 5 10 aaa ttt aac ctt ccg ccc ggg aat tac aaa aaa ccc aag ctt ctt tac 211 Lys Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr  15 20 25 30 tgc agt aac gga gga cac ttc ctg cga att ctg cca gat ggc aca gta 259 Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val                  35 40 45 gat ggg act cgc gat cgc tcc gac cag cac att cag ctg caa ctc tcg 307 Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser              50 55 60 gcc gaa agc gtt gga gag gtc tat atc aag tcg acg gag act ggc cag 355 Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln          65 70 75 tac ctt gcc atg gac acc gat ggg ctt ctg tat ggc tca cag acg cct 403 Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro      80 85 90 aac gaa gaa tgc ttg ttt cta gaa aga cta gaa gaa aac cat tac aac 451 Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn  95 100 105 110 acg tac ata tcg aaa aaa cat gca gag aag aac tgg ttt gta ggc ctt 499 Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu                 115 120 125 aaa aaa aat ggt tcc tgt aag cgt gga cca cgg act cac tat ggc caa 547 Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln             130 135 140 aag gct atc ttg ttc ctg cca cta cca gtg acg tcc gac taag 590 Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Thr Ser Asp         145 150 155 gatccgaatt cgagctccgt cgacaagctt gcggccgcac 630 <210> 2 <211> 155 <212> PRT <213> Homo sapiens <400> 2 Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe   1 5 10 15 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser              20 25 30 Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly          35 40 45 Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu      50 55 60 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu  65 70 75 80 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu                  85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr             100 105 110 Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys         115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala     130 135 140 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 <210> 3 <211> 468 <212> DNA <213> Homo sapiens <400> 3 atggctgaag gggaaatcac caccttcaca gccctgaccg agaagtttaa tctgcctcca 60 gggaattaca agaagcccaa actcctctac tgtagcaacg ggggccactt cctgaggatc 120 cttccggatg gcacagtgga tgggacaagg gacaggagcg accagcacat tcagctgcag 180 ctcagtgcgg aaagcgtggg ggaggtgtat ataaagagta ccgagactgg ccagtacttg 240 gccatggaca ccgacgggct tttatacggc tcacagacac caaatgagga atgtttgttc 300 ctggaaaggc tggaggagaa ccattacaac acctatatat ccaagaagca tgcagagaag 360 aattggtttg ttggcctcaa gaagaatggg agctgcaacg gcggtcctcg gactcactat 420 ggccagaaag caatcttgtt tctccccctg ccagtctctt ctgattaa 468 <210> 4 <211> 630 <212> DNA <213> Artificial Sequence <220> <223> This sequence is a chemically synthesized sequence encoding a 134          amino form of fibroblast growth factor with alterations for          preferred codon usage in E. coli. <221> CDS (222) (122) .. (526) <400> 4 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt 60 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca 120 t atg aat tac aaa aaa ccc aag ctt ctt tac tgc agt aac gga 163            Met Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly              1 5 10 gga cac ttc ctg cga att ctg cca gat ggc aca gta gat ggg act cgc 211 Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg  15 20 25 30 gat cgc tcc gac cag cac att cag ctg caa ctc tcg gcc gaa agc gtt 259 Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val                  35 40 45 gga gag gtc tat atc aag tcg acg gag act ggc cag tac ctt gcc atg 307 Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met              50 55 60 gac acc gat ggg ctt ctg tat ggc tca cag acg cct aac gaa gaa tgc 355 Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys          65 70 75 ttg ttt cta gaa aga cta gaa gaa aac cat tac aac acg tac ata tcg 403 Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser      80 85 90 aaa aaa cat gca gag aag aac tgg ttt gta ggc ctt aaa aaa aat ggt 451 Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly  95 100 105 110 tcc tgt aag cgt gga cca cgg act cac tat ggc caa aag gct atc ttg 499 Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu                 115 120 125 ttc ctg cca cta cca gtg agc tcc gac taag gatccgaatt cgagctccgt 550 Phe Leu Pro Leu Pro Val Ser Ser Asp             130 135 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa 610 caaagcccga aaggaagctg 630 <210> 5 <211> 135 <212> PRT <213> Homo sapiens <220> <223> Translated protein sequence for the chemically synthesized          134 amino acid form of fibroblast growth factor <400> 5 Met Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His   1 5 10 15 Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg              20 25 30 Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu          35 40 45 Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr      50 55 60 Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe  65 70 75 80 Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys                  85 90 95 His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys             100 105 110 Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu         115 120 125 Pro Leu Pro Val Ser Ser Asp     130 135 <210> 6 <211> 630 <212> DNA <213> Artificial Sequence <220> <223> Trasnslated protein sequence for the chemically synthesized          134 amino acid formo of fibroblast growth factor <221> CDS (122) .. (544) <400> 6 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt 60 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca 120 t atg ttt aac ctt ccg ccc ggg aat tac aaa aaa ccc aag ctt 163            Met Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu              1 5 10 ctt tac tgc agt aac gga gga cac ttc ctg cga att ctg cca gat ggc 211 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly  15 20 25 30 aca gta gat ggg act cgc gat cgc tcc gac cag cac att cag ctg caa 259 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln                  35 40 45 ctc tcg gcc gaa agc gtt gga gag gtc tat atc aag tcg acg gag act 307 Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr              50 55 60 ggc cag tac ctt gcc atg gac acc gat ggg ctt ctg tat ggc tca cag 355 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln          65 70 75 acg cct aac gaa gaa tgc ttg ttt cta gaa aga cta gaa gaa aac cat 403 Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His      80 85 90 tac aac acg tac ata tcg aaa aaa cat gca gag aag aac tgg ttt gta 451 Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val  95 100 105 110 ggc ctt aaa aaa aat ggt tcc tgt aag cgt gga cca cgg act cac tat 499 Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr                 115 120 125 ggc caa aag gct atc ttg ttc ctg cca cta cca gtg agc tcc gac 544 Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp             130 135 140     taagga tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc 600 accaccacca ctgagatccg gctgctaaca 630 <210> 7 <211> 141 <212> PRT <213> Homo sapiens <220> <223> Translated protein sequence for the chemically synthesized          140 amino acid form of fibroblast growth factor <400> 7 Met Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr   1 5 10 15 Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val              20 25 30 Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser          35 40 45 Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln      50 55 60 Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro  65 70 75 80 Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn                  85 90 95 Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu             100 105 110 Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln         115 120 125 Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp     130 135 140 <210> 8 <211> 1822 <212> DNA <213> Artificial Sequence <220> <221> TATA_signal (222) (102) .. (107) <221> CDS (222) (193) .. (201) <221> intron 202 (202) .. (457) <221> CDS <222> (458) .. (619) <221> intron <222> (620) .. (828) <221> CDS (222) (829) .. (948) <221> intron (222) (949) .. (1041) <221> CDS (222) (1206) <221> intron (222) (1207) .. (1459) <221> CDS (222) (1460) .. (1654) <223> Chemically synthesized sequence for Human Growth Hormone          using codons preferred for expression in E. coli <400> 8 ggagcttcta aattatccat tagcacaagc ccgtcagtgg ccccatgcat aaatgtacac 60 agaaacaggt gggggcaaca gtgggagaga aggggccagg gtataaaaag ggcccacaag 120 agaccggctc aaggatccca aggcccaact ccccgaacca ctcagggtcc tgtggacgct 180 cacctagctg ca atg gct aca ggtaagcgc ccctaaaatc cctttgggca 230                       Met ala thr                         One caatgtgtcc tgaggggaga ggcagcgacc tgtagatggg acgggggcac taaccctcag 290 gtttggggct tctgaatgag tatcgccatg taagcccagt atggccaatc tcagaaagct 350 cctggtccct ggagggatgg agagagaaaa acaaacagct cctggagcag ggagagtgct 410 ggcctcttgc tctccggctc cctctgttgc cctctggttt ctcccca ggc tcc cgg 466                                                        Gly ser arg                                                              5 acg tcc ctg ctc ctg gct ttt ggc ctg ctc tgc ctg ccc tgg ctt caa 514 Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu Cys Leu Pro Trp Leu Gln              10 15 20 gag ggc agt gcc ttc cca acc att ccc tta tcc agg ctt ttt gac aac 562 Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn          25 30 35 gct atg ctc cgc gcc cat cgt ctg cac cag ctg gcc ttt gac acc tac 610 Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr      40 45 50 cag gag ttt g taagctcttg gggaatgggt gcgcatcagg ggtggcagga 660 Gln glu phe  55 aggggtgact ttcccccgct gggaaataag aggaggagac taaggagctc agggtttttc 720 ccgaagcgaa aatgcaggca gatgagcaca cgctgagtga ggttcccaga aaagtaacaa 780 tgggagctgg tctccagcgt agaccttggt gggcggtcct tctcctag gaa gaa gcc 837                                                        Glu glu ala                                                                 60 tat atc cca aag gaa cag aag tat tca ttc ctg cag aac ccc cag acc 885 Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr                  65 70 75 tcc ctc tgt ttc tca gag tct att ccg aca ccc tcc aac agg gag gaa 933 Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu              80 85 90 35 aca caa cag aaa tcc gt gagtggatgc cttgacccca ggcggggatg 980 Thr Gln Gln Lys Ser          95 ggggagacct gtagtcagag cccccgggca gcacaggcca atgcccgtcc ttcccctgca 1040 g aac cta gag ctg ctc cgc atc tcc ctg ctg ctc atc cag tcg 1083            Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser                    100 105 110 tgg ctg gag ccc gtg cag ttc ctc agg agt gtc ttc gcc aac agc ctg 1131 Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu             115 120 125 30 gtg tac ggc gcc tct gac agc aac gtc tat gac ctc cta aag gac cta 1179 Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu         130 135 140 gag gaa ggc atc caa acg ctg atg ggg gtgg gggtggcgct aggggtcccc 1230 Glu Glu Gly Ile Gln Thr Leu Met Gly     145 150 aatcttggag ccccactgac tttgagagct gtgttagaga aacactgctg ccctcttttt 1290 agcagtccag gccctgaccc aagagaactc accttattct tcatttcccc tcgtgaatcc 1350 tctagccttt ctctacaccc tgaaggggag ggaggaaaat gaatgaatga gaaagggagg 1410 gagcagtacc caagcgcttg gcctctcctt ctcttccttc actttgcag agg ctg gaa 1468                                                        Arg Leu Glu                                                                155 gat ggc agc ccc cgg act ggg cag atc ttc aag cag acc tac agc aag 1516 Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys                 160 165 170 ttc gac aca aac tca cac aac gat gac gca cta ctc aag aac tac ggg 1564 Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly             175 180 185 ctg ctc tac tgc ttc agg aag gac atg gac aag gtc gag aca ttc ctg 1612 Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu         190 195 200 cgc atc gtg cag tgc cgc tct gtg gag ggc agc tgt ggc ttc tagctg 1660 Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe     205 210 215 cccgggtggc atccctgtga cccctcccca gtgcctctcc tggccttgga agttgccact 1720 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg 1780 tcctctataa tattatgggg tggagggggg tggtttggag ca 1822 <210> 9 <211> 217 <212> PRT <213> Homo sapiens <400> 9 Met Glu Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu   1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu              20 25 30 Ser Arg Leu Phe Glu Asn Ala Met Leu Arg Ala His Arg Leu His Gln          35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys      50 55 60 Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe  65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys                  85 90 95 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp             100 105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val         115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu     130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser                 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe             180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys         195 200 205 Arg Ser Val Glu Gly Thr Cys Gly Phe     210 215 <210> 10 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Chemically synthesized seqeuence for human interferon alpha-2b <221> promoter (231) .. (249) <221> CDS (222) (320) .. (784) <400> 10 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt 60 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg 120 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag 180 gagcacgatc atgcgcaccc gtggggccgc cagatctcga tcccgcgaaa ttaatacgac 240 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact 300 ttaagaagga gatatacat atg gct gaa ggg gaa atc acc acc ttt aca gcg 352                       Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala                         1 5 10 tta acg gag aaa ttt aac ctt ccg ccc ggg aat tac aaa aaa ccc aag 400 Leu Thr Glu Lys Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys              15 20 25 ctt ctt tac tgc agt aac gga gga cac ttc ctg cga att ctg cca gat 448 Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp          30 35 40 ggc aca gta gat ggg act cgc gat cgc tcc gac cag cac att cag ctg 496 Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu      45 50 55 caa ctc tcg gcc gaa agc gtt gga gag gtc tat atc aag tcg acg gag 544 Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu  60 65 70 75 act ggc cag tac ctt gcc atg gac acc gat ggg ctt ctg tat ggc tca 592 Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser                  80 85 90 cag acg cct aac gaa gaa tgc ttg ttt cta gaa aga cta gaa gaa aac 640 Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn              95 100 105 cat tac aac acg tac ata tcg aaa aaa cat gca gag aag aac tgg ttt 688 His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe         110 115 120 gta ggc ctt aaa aaa aat ggt tcc tgt aag cgt gga cca cgg act cac 736 Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His     125 130 135 tat ggc caa aag gct atc ttg ttc ctg cca cta cca gtg agc tcc gac 784 Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 140 145 150 155     taagga tccgaattcg agctccgtcg acaagcttgc ggccgcactc gacgaccacc 840 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg 900 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt 960 ttttgctgaa aggaggaact atatccggat 990 <210> 11 <211> 155 <212> PRT <213> Homo sapiens <220> <223> Translated protein sequence for the chemically synthesized          human interferon alpha-2b <400> 11 Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe   1 5 10 15 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser              20 25 30 Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly          35 40 45 Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu      50 55 60 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu  65 70 75 80 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu                  85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr             100 105 110 Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys         115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala     130 135 140 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155

Claims (24)

Transforming an E. coli strain capable of expressing a gene of a T7 RNA polymerase with a plasmid having at least one copy of an expressible gene encoding a bioactive protein linked to operate on a T7 polymerase promoter; Infecting said transformed bacterial host cell with a bacteriophage having a mutation in one or more genes selected from the group consisting of N, Q, and R that can control delayed lysis; And Culturing E. coli host cells until soluble and bioactive proteins are produced in desired levels. The method of claim 1, wherein the bacteriophage has a temperature sensitive mutation cl ₈₅₇ . delete The method of claim 2, wherein before the culturing step, the E. coli host cells are grown at a temperature that prevents lytic growth of the bacteriophage. delete delete The method of claim 1, wherein the E. coli strain produces an inhibitory factor for modifying amber mutation. The method of claim 1, wherein the E. coli strain is free of inhibitory factors to modify amber mutation. The method of claim 1, wherein said infectious bacteriophage is provided in a multiplicity of infection in the range of 1-10. The method of claim 1, wherein said infectious bacteriophage is provided at a multiplicity of infection in the range of 10-25. The method of claim 1, wherein the bacteriophage-regulated delayed dissolution of the E. coli strain is delayed at high multiplicity of infection compared to low multiplicity of infection. The method of claim 1, wherein said expressible gene encodes an acidic fibroblast growth factor of humans. 13. The method of claim 12, wherein the acidic fibroblast growth factor of humans comprises 134 amino acids. 13. The method of claim 12, wherein said acidic fibroblast growth factor of humans comprises 140 amino acids. 13. The method of claim 12, wherein said acidic fibroblast growth factor of humans comprises 146 amino acids. The method of claim 12, wherein said acidic fibroblast growth factor of humans comprises 155 amino acids. The method of claim 16, wherein said acidic fibroblast growth factor of human has the sequence shown in SEQ ID NO: 1. The method of claim 1, wherein said expressible gene encodes a human growth hormone. The method of claim 1, wherein the expressible gene encodes human interferon. The method of claim 1, wherein the expressible gene encodes E. coli methionine amino peptidase. The method of claim 1, wherein the gene of the T7 RNA polymerase is under the control of an inducible promoter. The method of claim 21, wherein the inducible promoter is a lac UV 5 promoter. A chemically synthesized nucleic acid consisting of the sequence shown in SEQ ID NO: 1. a) growing the first strain of E. coli living bacteriophage λ strain having a temperature-sensitive mutation cl ₈₅₇ ; b) adjusting the temperature to dissolve the first strain of E. coli and release the bacteriophage? c) under the control of an inducible promoter, transformed into a plasmid having at least one copy of an expressable gene encoding a physiologically active protein operably linked to a T7 polymerase promoter, and adding an inducer to the gene of the T7 RNA polymerase. Providing a second strain of E. coli that can be induced to express; d) infecting the second strain of E. coli with the bacteriophage λ released from the first strain of E. coli; And e) the infected E. coli agent in a culture medium containing an inducing factor such that soluble, bioactive proteins are produced at a concentration higher than 100 μg / ml and released into the culture medium upon dissolution of the second strain of E. coli. A method of producing a bioactive protein comprising the step of culturing for two weeks.

Images